The long term goal of this project is to develop a quantitative understanding of how individual interactions contribute cooperatively to conformational stability in both fully folded proteins and in partially folded intermediates that determine the mechanism of folding. Amino acid replacements will be used to probe the roles of individual residues in the refolding of bovine pancreatic trypsin inhibitor (BPTI), a small protein for which the folding pathway has been studied in great detail. Disulfide- bonded intermediates in the refolding of this protein contain much of the structure found in the native protein, but the extent and stability of this structure varies among the intermediates. Measuring the effects of amino acid replacements on the stabilities of the different intermediates will provide a uniquely detailed picture of how the contributions of individual residues are influenced by their environment within the protein at different stages of folding. Genetically modified BPTI variants will be produced in Escherichia coli and will be studied by chemically trapping, isolating and characterizing the disulfide-bonded intermediates, using techniques previously developed for the study of the wild-type protein. One of the major goals of this project is to measure the effects of substitutions on the kinetics and equilibria for forming the intermediates that contain only a single disulfide and represent an early stage of folding. Comparing the effects of substitutions on these intermediates with the effects of the same substitutions on the stability of the native protein will identify the major interactions that stabilize the intermediates and will indicate whether these interactions are as stable in the intermediates as they are in the fully folded protein. The second major goal is to learn how the stability of a single interaction in the native protein, a disulfide bond, is influenced by its environment For this purpose, the effects of substitutions on the last step of the pathway, the formation of a disulfide in the otherwise native protein, will be studied. Single amino acid replacements can alter the free energy change for this reaction by as much as 5 kcal/mol. To determine the structural basis of these effects, high resolution NMR will be used to study the structures and dynamics of mutant proteins with and without the disulfide. In addition, the roles of steric and entropic factors in determining disulfide stability will be probed by measuring the effects of multiple amino acid replacements and temperature on the equilibria and kinetics of the reaction. The results of this project will help provide the fundamental knowledge necessary to fully realize the health care potentials offered by the revolutionary developments in genetic analysis and manipulation. Possible applications of this knowledge include the design of proteins with enhanced stabilities or novel activities and improved understanding of how naturally occurring mutations lead to disease states by altering protein structure and dynamics.